Proteoglycans are molecules that contain both a protein portion (which may be referred to as the protein core) and glycosaminoglycan portion. Glycosaminoglycans are the most widely present polysaccharides in the animal kingdom and are mainly found in the connective tissues. Glycosaminoglycans are biological polymers made up of linear disaccharide units containing an uronic acid and a hexosamine and are attached to the core proteins via a linking tetrasaccharide moiety. The major glycosaminoglycans are hyaluronic acid, chondroitin sulfates, heparan sulfate, dermatan sulfate and keratan sulfate.
Physiochemical properties such as, but not limited to, solvation and interaction with biological tissues and structures are related to, at least in part, the presence of carboxylic acid and/or sulfuric groups within the glycosaminoglycan chains. The glycosaminoglycan chains can impart a substantial negative charge to the proteoglycan proteins. The aligned sulfated glycosaminoglycans chains can sequester water through hydrogen bonding, holding many times their weight in water. Such properties determine, in part, the biological activity of the glycosaminoglycans in extracellular matrices. Glycosaminoglycans also help to maintain a favorable environment for cell growth. Because of these characteristics, glycosaminoglycans are used in many clinical and medical applications. The use of glycosaminoglycans in cosmetics is also due to their physicochemical and biological properties linked to their molecular weight and their water retaining capacity (Gagnieu, et al., Peau Seche et Hydratation, Lyon, France, Journees Internationales de Dermocosmetologie de Lyon, 1998). The hydrating potential of recombinantly expressed proteoglycans has also been published (Huc, et al., Peau Seche et Hydration, Lyon, France, 1988).
Proteins that are membrane bound become either integral membrane proteins, with a transmembrane domain, or glycosylphosphotidylinositol (GPI) linked to the lipid bilayer. Within an integral membrane protein, a transmembrane helix is a segment that is alpha-helical in structure, roughly 20 amino acids in length, and is said to “span” the membrane.
Glycosaminoglycan chains are synthesized via similar routes involving the stepwise addition of four monosaccharides to serine residues followed by co-polymerization of disaccharide units. It has been shown that serine followed by glycine residues are heavily favored acceptors for xylosyltransferase, the key enzyme in the initial step of glycosaminoglycan glycosylation. The priming of glycosaminoglycan synthesis as well as determining the type of glycosylation is in part encoded by the core protein. The formation of heparan sulfate over chondroitin sulfate, for example, is favored when the core protein contains 2 or more serine-glycine (SG)-consensus sequences in close proximity to each other, a cluster of acidic amino acids is located nearby, and a tryptophan residue immediately follows the (SG) glycosaminoglycan attachment sites. However, acidic clusters are also found in chondroitin sulfate proteoglycans and thus seem to be necessary but not sufficient for the priming of heparan sulfate glycosylation. Therefore, while the sites for glycosaminoglycan attachment are known to some extent, the rules governing the type and extent of glycosaminoglycan modification are still not clear. The GAG attachment sites are known for a number of proteoglycans, including perlecan (Dolan et al, J. Biol. Chem., Vol. 272, p. 4316-4322, 1997), collagen XVIII (Dong et al., J. Biol. Chem., Vol. 278, p. 1700-1707, 2003), syndecan-1 (Zhang et al., J. Biol. Chem., Vol. 270, p. 27127-27135, 1995), glypican (Chen et al., J. Biol. Chem., Vol. 276, p. 7507-7517, 2001) and agrin (Winzen et al., J. Biol. Chem., Vol. 278, p. 30106-30114, 2003).
Chronic sun exposure induces numerous changes in exposed skin; the most striking histopathologic change is the massive accumulation of material with the staining characteristics of elastin, termed solar elastosis, in the superficial dermis. Recently, the large chondroitin sulfate proteoglycan, versican, has been identified in the dermis in association with elastic fibers, and the smaller chondroitin sulfate proteoglycan, decorin, has been shown to codistribute with collagen fibers. Evidence was published for the close association of versican with elastic fibers and decorin with collagen fibers, even in the situation of abnormal fiber deposition occurring in photodamaged skin (Bernstein, et al., Lab Invest 72(6): 662-9, 1995). In addition, changes in versican and decorin immunostaining were accompanied by similar alterations in gene expression. In another study, the effect of UVB exposure on the distribution and synthesis of dermal proteoglycans was measured in the skin of mice (Margelin, et al., Photochem Photobiol 58(2): 211-8, 1993). The results demonstrated that chronic doses of UVB altered proteoglycan metabolism through both quantitative and qualitative changes.
Numerous articles have been published about the necessity of moisturizers for patients with dermatological damage, such as dryness caused by rosacea, and the ability of moisturizers to hydrate the skin and reduce transepidermal water loss (TEWL), further improving the vigor of the stratum corneum (Draelos, Dermatol Clin 18(4): 597-607, 2000; Bikowski, Cutis 68(5 Suppl): 3-11, 2001; Rawlings, et al., Dermatol Ther 17 Suppl 1: 49-56, 2004; Draelos, Cutis 76(6 Suppl): 19-25, 2005). Such moisturizers may contain proteoglycans. Although these moisturizers have been shown to improve patient skin health and appearance, they are presently only delivered as pre-synthesized proteoglycans and so limited in their effectiveness.
Despite the widespread use and acceptance of proteoglycans and glycosaminoglycans in pharmaceuticals, the use of proteoglycans when delivered as polynucleotides has only been described for the proteoglycans decorin and biglycan in the scientific literature, and for decorin (U.S. Pat. No. 6,524,573) and perlecan (U.S. Pat. No. 7,141,551) in the patent literature. In addition, a patent has been issued describing in-situ delivery of nucleic acid encoding syndecan intracellular sequences as a fusion transmembrane protein that is expressed as a cell-bound molecule (U.S. Pat. No. 7,029,668). This is substantially different in scope and meaning from the present disclosure, which discloses a polynucleotide encoding a secreted proteoglycan. In managing skin homeostasis, secreting the proteoglycan into the extracellular matrix is an important aspect of the present invention and has not been demonstrated as obvious to experts in the field.
Due to their propensity to sequester water, the delivery of proteoglycan polypeptides to the sites of wounds or cutaneous injury caused by age, sun exposure, skin ailments, or trauma will visibly reduce wrinkling or surface irregularities as the sequestered water hydrates the area and cause conditions favorable for treatment and/or prevention of wounds and/or cutaneous injury. In addition, more highly hydrated skin should function better, possibly allowing for increased blood flow and elasticity, further improving appearance, and increasing patient health by allowing nutrients to be distributed more readily.
The present disclosure provides nucleic acid constructs encoding one or more polypeptides containing one or more sites for glycation by glycosaminoglycan chain, such as proteoglycan polypeptides, and methods for delivering to the site of a wound or cutaneous injury at least one nucleic acid construct encoding one or more such polypeptides, such that the expressed polypeptide is decorated by glycosaminoglycan chains through the normal physiological processes of the subject at the site of administration to produce a polypeptide for the healing of the wound or other cutaneous injury. The delivered nucleic acid construct is transcribed, translated to produce a polypeptide, which is subsequently post-translationally modified by the addition of glycosaminoglycan chains (referred herein as “decoration” or “glycation”) to produce a polypeptide. The glycated polypeptide is then secreted from the cell in which it was produced as directed by a sequence that is sufficient for secretion. During the secretion process, all or a portion of such sequence may be cleaved. After secretion, the glycated polypeptide is available to provide treatment of wounds and/or cutaneous injury in the subject and/or prevention of cutaneous injury in a subject. As a result, the glycated polypeptide released is decorated optimally by the normal physiological process of the subject to provide one or more glycosaminoglycan containing polypeptides based on the conditions prevailing at the site of administration in a particular subject. In a particular embodiment, the glycosaminoglycan containing polypeptide is a proteoglycan polypeptide.